Sulphated hyaluronic acids as regulator agents of the cytokine activity

ABSTRACT

The object of the present invention relates to the new and surprising use of sulfated hyaluronic acid (HAS) as regulator agent of the cytokine activity (pro- and antiinflammatory) and consequently the use of HAS for the preparation of a new medicine for the prevention and treatment of pathologies associated with the activation and/or deficiency of cytokines of a pro- and antiinflammatory nature. The Applicant has in fact discovered its exclusive capacity in modulating the activity of these particular proteins, it has studied the action mechanism and demonstrated the substantial difference between the different sulfated types known in the state of the art, but above all it has demonstrated an unexpectedly high activity of HAS vs different types and strains of Herpes virus, HIV, Cytomegalovirus and the virus of vesicular stomatitis.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a Divisional of copending application Ser. No.13/320,015, filed on Jan. 4, 2012, which was filed as PCT InternationalApplication No. PCT/EP2010/003044 on May 11, 2010, and under 35 U.S.C. §119(a) to Patent Application No. PD2009A 000135, filed in ITALY on May14, 2009, all of which are hereby expressly incorporated by referenceinto the present application.

FIELD OF THE INVENTION

For many years now, scientific/patent literature has been studying anddescribing sulfated hyaluronic acid which is obtained starting fromhyaluronic acid (HA) suitably sulfated according to what is described inthe state of the art (EP0940410B1 and EP0702699B1), to whichanticoagulant effects are attributed. HAS can also be obtained by thede-acetylation and subsequent sulfation of glucosamine of HA (defined asHA-NS) (EP0971961B1), for the production of surgical articles andpharmaceutical compositions. Patents EP0754460B1 and EP1385492B1 arealso known, in which the use of HAS is described in pathologies such as,for example, ARDS, articular rheumatism, rheumatoid arthritis anddermatitis.

An object of the present invention relates to the new and surprising useof HAS as regulator agent of the cytokine activity, as the Applicant hasdiscovered the exclusive capacity of HAS of modulating the activity ofparticular cytokines (both pro- and anti-inflammatory), it has studiedits action mechanism and revealed the substantial difference between thetwo types of sulfated product (HAS and HA-NS), but above all theApplicant has surprisingly found an unexpectedly high activity vsdifferent types and strains of Herpes virus, HIV, Cytomegalovirus andthe virus of vesicular stomatitis.

Since 1970, scientists have understood that selected populations oflymphoid cells can produce and release into the circulatory bed,molecules of a protein nature not assimilable to antibodies, definedwith the term “cytokines”. They represent a new type of “hormone”,capable of acting on different cell targets in numerous regions of thebody.

The progression of scientific knowledge relating to the synthesis andbiological/biochemical functions of these proteins, has altered the“old” vision of the immune system (I.S.) of the same scientific worldand has opened up new horizons in understanding its numerous functions,thus creating new prospects for the treatment of different pathologies,topic and/or systemic, also comprising new therapeutic possibilitiesrelating to the immunotherapy of cancer.

The central IS cell is the lymphocyte, it represents about 20% of allthe white blood cells and, on the basis of its various functions, forms3 groups: lymphocyte B, lymphocyte T and killer lymphocyte. Manycytokines are soluble proteins produced by lymphocytes and/or monocytes,capable of acting against other cells/tissues also situated very farfrom their production site. They have immunological functions, in fact,and also regulation functions in the synthesis of other cytokines bydifferent cells of the I.S. or target cells involved in the cascade ofreactions initiated by the I.S.

Numerous different cytokines have been studied so far, also havingnumerous different acronyms, but those studied in particular by theApplicant are: Interleukin 1 and 2, Interleukin 6, 7 and 12, hereafterdefined as IL-1, IL-2, IL-6, IL-7 and IL-12 which, with TNF, are definedas cytokines of a pro-inflammatory nature, whereas Interleukin 10(IL-10) on the contrary, is a cytokine with strong anti-inflammatoryproperties.

The first cytokine to be studied was definitely IL-1 present in twoforms α and β, a powerful inducer of inflammatory processes, both localand systemic. It is mainly produced by lymphocytes B, T and macrophagesafter bacterial stimulus or stimulation on the part of other agentsincluding other cytokines; it is also secreted from alveolar, peritonealmacrophages, Kupffer cells, peripheral neutrophils, endothelial,epithelial and smooth muscle cells, fibroblasts, Langerhans cells of theskin, osteoclasts, synoviocytes and many other types of cell. It is alsopresent in the cerebrospinal fluid where it is both produced andtransported. Both forms bind the same receptor and have very similar, ifnot identical, biological activities. Many of their pro-inflammatoryfunctions are linked to the stimulation of other cytokines, such as IL-6and IL-8, and their very synthesis can be induced by cytokines such asTNF, Interferon, bacterial endotoxins, viruses and different types ofother antigens. It is involved in septic shock but it should also benoted that recent studies have demonstrated that IL-1 is capable ofactivating the expression of some oncogenes and consequently ofparticipating in the pathogenesis of neoplasias. Furthermore, anautocrin control system of the growth of blast cells of the white serieswhich are present in the circulatory bed of patients with the leukaemiapathology has been suggested for this cytokine: these blast cells infact uncontrollably produce IL-1 which in turn stimulates the synthesisof those growth factors which increase the proliferation of the sameblast cells. Combined with other cytokines, IL-1 therefore representsone of the major mediators of inflammatory processes: it stimulatesT-cells in fact to produce IL-2 and B-cells to produce immunoglobulins.It is consequently involved in numerous pathologies such as, forexample, astrogliosis and the demyelination of neuronal fibres, it iscytotoxic for Langerhans pancreatic cells which produce insulin, and isalso involved in lithic processes of the bones, both activatingosteoclasts and suppressing the formation of new bone, both processesbeing involved in the pathology of osteoporosis. It is capable offunctioning as an endogenous pyrogen as, by increasing the release ofprostaglandins in the hypothalamic centre, it causes an increase in thebody temperature. It is also involved in the pathogenesis of rheumatoidarthritis and arthrosis: high quantities of IL-1 have in fact been foundin the synovial fluid of patients affected by rheumatoid arthritisand/or osteoarthrosis. Finally, it participates in the establishment ofvascular damage such as venous thrombosis and is present in all vesselswith pathologies of the arterio/arterioschlerotic type. Receptorantagonists are currently being experimented for this cytokine, asblockage of the receptor is proving to be an effective way of treatingthese pathologies in which IL-1 is among the protagonists.

TNF: The necrosis factor is part of the group of cytokines whichpromotes the reaction of acute systemic inflammation phase. TNF istherefore involved in an extremely wide number of processes such as cellproliferation, differentiation and apoptosis, carcinogenesis and viralreplication.

It is mostly produced by macrophages and by a series of other cell typesincluding mastocytes, lymphoid cells, muscular and endothelial cells,fibroblasts and nerve cells. Its synthesis can be stimulated bybacterial endotoxins, other cytokines such as IL-2, Interferon and IL-1,and it can be inhibited by steroids.

By acting on numerous organs and systems, generally together with othercytokines, it participates in the establishment and regulation of manypathogenetic processes:

-   -   it modulates the expression of many proteins and important        cytokines, such as IL-1 and IL-6 thus resulting involved in many        cutaneous pathologies such as, dermatitis, vitiligo and eczema;    -   it stimulates the hypothalamo-hypophyseal-adrenal axis        increasing the release of some types of hormones;    -   it suppresses the appetite;    -   it induces fever (it acts as endogenous pyrogen inducing the        hypothalamus to produce prostaglandins);    -   it stimulates the synthesis of collagenasis in the synoviocytes        and for this reason, large quantities of TNF have been found in        the synovial fluids of patients suffering from arthrosis and        rheumatoid arthritis;    -   it activates the osteoclasts and therefore induces re-absorption        of the bone, process characterizing the osteoporosis pathology;    -   it is also involved in pathologies of the Nervous System, such        as astrocytosis and demyelination;    -   it strongly attracts neutrophils and helps them attach        themselves to the endothelial cells to extravasate;    -   it stimulates the macrophagic production of molecules with an        oxidizing action;    -   it is involved in particular pathologies of the        Cardio-circulatory System participating in the formation of        venous thrombosis, in the pathogenesis of arteriosclerosis and        vasculitis;    -   it increases the resistance to insulin, it increases the protein        catabolism in the muscles whereas it suppresses the lipogenic        metabolism in the adipose tissue.

High concentrations of TNF can induce shock-like symptoms, whereasprolonged exposure to low concentrations can lead to cachexia, asyndrome which causes the depletion of the protein and lipid patrimonyof the tissues (in particular muscular and adipose). The TNF is capableof binding itself to two receptors, TNF-R1 (receptor for type 1 TNF) andTNF-R2 (receptor for type 2 TNF), which are expressed in all somaticcells excluding erythrocytes. In short, the TNF promotes theinflammatory response which in turn triggers numerous pathologies alsoof an auto-immune nature, such as rheumatoid arthritis, Crohn disease,psoriasis and asthma. Scientific research has so far tried to perfect“biological” drugs (such as, for example, monoclonal antibodies) whichinhibit the synthesis of TNF and/or block its receptor.

IL-2: this is a highly pro-inflammatory, atherogenic cytokine, mainlyproduced by lymphocytes T, whose synthesis is inhibited by steroids andcyclosporines. Leukemic cells synthesize the above cytokine andcontemporaneously express its receptor, thus creating an autocrin systemin the stimulation of their growth which causes a deterioration in theleukemia pathology. IL-2 has a central role in regulating theimmunological response: it stimulates in fact the synthesis of IFN inthe peripheral leukocytes and induces the production of IL-1 and TNF.IL-2 can also damage the hematoencephalic barrier and integrity of theendothelium of the cerebral vessels, causing neuropsychiatric disorderssuch as disorientation and depression.

There are consequently numerous pathologies which have been associatedwith an aberrant production of IL-2, such as Hodgkin's lymphoma, therejection of organ transplants, multiple sc(h)lerosis, rheumatoidarthritis, Lupus erythematosus, diabetes and AIDS.

IL-6: produced by many cell types above all of I.S., with TNF it isamongst the most important members of the group of chemical mediators ofthe acute phase of the inflammatory process, and is therefore involvedin pathologies with a strong inflammatory component, such as asthma(where it participates in the emergence and maintenance of theinflammatory process), chronic intestinal inflammation (Crohn'sdisease), rheumatoid arthritis and arthrosis. As previously affirmed, infact, cytokines such as TNF, IL-1 and IL-6 have proved to be greatlyinvolved in the degenerative articular osteoarthrosis process as theyhave a primary role in regulating the expression of metalloproteases(responsible for cartilage degradation), in the production ofprostaglandins and in osteoclastic activation and, for this reason, highcytokine levels have been registered in the synovial fluids of patientssuffering from arthrosis and rheumatoid arthritis (R.A.). Thesediscoveries have stimulated the use of inhibitors in the aboveinterleukins and/or receptor antagonists as a new treatment strategy ofthe arthrosis pathology.

High concentrations of IL-6 have also been found in the urine ofpatients subjected to transplants and its presence represents one of thefirst signs of the rejection reaction of the organ. The serum level ofthis cytokine is also drastically increased in many patients sufferingfrom tumors (such as, for example, myeloma, leukemia, cardiac myxomas,or in pathologies such as lymphadenopathies and hepatic cirrhosis) andcan also be used as indicator in monitoring the size of the tumoralmass. Finally, recent studies have connected cancer with longevity andrevealed how some tumors are influenced by the quali/quantitativesituation of the cytokine proteins of the patient: in short, recentevidence has linked a low production profile of IL-10 and high secretionof IL-6 to a deterioration in the clinical survival of patients affectedby tumoral pathologies, whereas a genotype capable of producing andmaintaining high levels of IL-10 can facilitate survival. Consequently,persons with high antiinflammatory cytokine levels and a lowconcentration of pro-inflammatory cytokines are genetically susceptibleto having a greater longevity (Caruso C. et al., Ann N.Y. Acad. SCI.,2004, 1028:1-13).

IL-7: cytokine mainly produced by stromal cells of bone marrow, it isalso secreted by the thymus and keratinocytes. IL-7 induces thesynthesis of inflammatory cytokines such as IL-1, IL-6 and TNF, thusparticipating in the pathogenesis of some skin diseases (such aspsoriasis and cutaneous lymphoma) and the osteoarticular system. Highlevels of IL-7 have in fact been found in patients suffering from R.A.as IL-1 and TNF (cytokines strongly involved in the above pathology) canincrease the stromal production of IL-7 which in turn stimulates themacrophagic synthesis of TNF. Finally, IL-7 can induce the maturation ofosteoclasts and consequently increase bone resorption thus contributingto the degeneration of the joints.IL-12: this protein also plays a central role in regulating thefunctions of the I.S. It acts in fact on the differentiation of thelymphocytes, it induces the synthesis of Interferon and TNF, and itsproduction can be inhibited by IL-10. The over-production of thisprotein enters in the pathogenesis of diseases of (an) auto-immunenature such as colitis, arthritis, insulin-dependent diabetes,encephalomyelitis, psoriasis and multiple schlerosis (Brahmachari S. etal., Minerva Med., 2008, 99(2):105-118).IL-10: mainly produced by lymphocytes, it is a cytokine of ananti-inflammatory nature, capable of inhibiting the synthesis of IL-2and Interferon produced by lymphocytes T. The anti-inflammatory actionof IL-10 is also revealed in the capacity of inhibiting the synthesis ofIL-1, IL-6, IL-8, IL-12 and TNF in the macrophages stimulated withbacterial endotoxins. IL-10 deficiencies are associated with pathologiessuch as diabetes mellitus and chronic intestinal inflammations, such asCrohn's disease. Recent evidence has led IL-10 to also be experimentedas a new therapeutic approach for the treatment of Lupus erythematosus.Low IL-10 levels have been observed in cutaneous tissues of patientssuffering of pathologies such as vitiligo, psoriasis, eczema anddermatitis in general. It should be noted that both corticosteroids andcyclosporine increase the production and/or release of this interleukinfrom the relative competent cells during conventional immunosuppressiontherapy for the treatment of inflammations and organ rejection (Zhou X.Et al., Current Drug Targets-Immune, Endocrine & Metabolic Disorders,2005, 5(465475). Experimental data have also demonstrated itseffectiveness in reducing the release of prostaglandins andcyclo-oxygenase induced in vitro by TNF on human synoviocytes, thusindicating the capacity of IL-10 of reducing inflammatory processeswhich involve articulations affected by osteoarthrosic degeneration(Alaaeddine N. et al., Arthritis & Rheumatism, 1999, 42:710-718). Recentstudies have confirmed its therapeutic effectiveness towards the asthmapathology in experimental animal models of bronchial hyper-reactivity,showing how this cytokine has a high therapeutic potentiality inreducing the inflammation which characterizes the air passages ofasthmatic patients, in which high concentrations of TNF, IL-1, IL-5,IL-6 and IL-8 have been found in the bronchial-washing liquid and/or ona serum level and/or tissue level (Stankiewicz W. et al., Mediators ofInflammation, 2002, 11:307-312). For this interleukin, the importantrole of regulator cytokine of the maintenance of immunologicalhomeostasis, has therefore been assumed.

Asthma can be an extremely invalidating disease of which approximately200 million people in the world suffer, with over 5,000 deaths per year.It is a pathology which is based on a distorted response of the I.S. toenvironmental factors, consequently linked to an exacerbated productionof pro-inflammatory cytokines for the growth and differentiation of mastcells and eosinophils with other types of cells of the I.S. The causesof this out-of-balance activity of the immune system are still notcompletely known, there are however genetic, environmental, viral andalso nutritional factors which contribute in different ways to thedevelopment of this pathology. Consequently, finding an effectivetherapy for its prevention and/or treatment which allows the suspensionor reduction of the use of steroids (conventional treatment therapy),could represent a valid solution for both the more serious forms (as itwould in any case enable a reduction in the use of steroids) and forless serious cases, as the suspension of the steroid therapy could betotal.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the gene expression of IL-10 in human synoviocytes treatedwith HA, HAS1 or HAS3.

FIG. 2 shows the gene expression of IL-12 in human synoviocytes treatedwith HA, HAS1 or HAS3.

FIG. 3 shows binding inhibition percent of TNF with HA, HAS1 or HAS3.

FIG. 4 shows HAS3 binding inhibition percent of IL-1.

FIG. 5 shows HAS3 binding inhibition percent of IL-6.

FIG. 6 shows the synthesis of IL-2 in human PBMC treated with HA, HAS1or HAS3.

FIG. 7 shows the synthesis of IL-7 in human PBMC treated with HA, HAS1or HAS3.

FIG. 8 shows the synthesis of IL-12 in human PBMC treated with HA, HAS1or HAS3.

FIG. 9 shows the synthesis of IL-10 in human PBMC treated with HA, HAS1or HAS3.

FIG. 10 shows the antiviral activity against HIV strains of HA-NS1,HAS1, and HAS3.

FIG. 11 shows the antiviral activity against various herpes simplexstrains and vesicular stomatitis virus of HA-NS1, HAS1, and HAS3.

FIG. 12 shows the antiviral activity against cytomegalovirus strains ofHA-NS1, HAS1, and HAS3.

DETAILED DESCRIPTION OF THE INVENTION

An object of the invention is the new and surprising use of HAS asregulator agent of the cytokine activity, as the Applicant hasdiscovered its exclusive capacity in modulating the activity ofparticular cytokines, it has studied its action mechanism and revealedthe substantial difference between the various types of sulfatedproducts known in the state of the art, but above all the Applicant hasdiscovered an unexpected activity vs different types and strains ofHerpes virus, HIV, Cytomegalovirus and the virus of vesicularstomatitis. The sulfated hyaluronic acid suitable for the purposes ofthe present invention is prepared according to the process described inEP 702699 B1: the sulfation is effected by means of the complexSO₃-pyridine and involves the alcohol hydroxyls present in thepolysaccharide chain starting from a HA deriving from any source, forexample, by extraction from cockscombs, either fermentatively ortechnologically, and having a molecular weight ranging from 400 to 3×10⁶Da, in particular from 1×10⁴ Da to 1×10⁶ Da, even more in particularfrom 10,000 to 50,000 Da, 150,000 to 250,000 Da and 500,000 to 750,000Da.

The derivative obtained maintains all the physical characteristics ofthe starting polymer unaltered, in particular the molecular weight ofthe starting HA is not altered by the sulfation process thus allowingall the physico-chemical characteristics of the starting polysaccharideto be maintained. The sulfation involves the various hydroxyl groups ofthe disaccharide unit and it is therefore possible to obtain differentsulfation degrees, from 0.5 to 3.5 (intended as the number of sulfategroups per disaccharide unit), by varying the quantity of SO₃-pyridineintroduced as known in the state of the art.

The derivative used in all the experimentations effected generally hassulfation degree 1 or degree 3 and is defined hereafter as HAS1 andHAS3. All the free carboxyl groups of the HA can be salified withcations of an organic and/or inorganic origin.

Both degrees of HAS are soluble in water and they can also be sterilizedwith the normal techniques known to experts in the field, even ifsterilization using an autoclave is preferable.

In the experimentations described hereunder, the Applicant demonstratedthat:

-   -   HAS is capable of both stimulating the production of new mRNA        and the protein synthesis of anti-inflammatory cytokines (such        as, for example, IL-10), thus increasing the immune defense        capacity of the cells and therefore of the whole organism. The        anti-inflammatory action of the above cytokines is revealed in        the capacity of inhibiting the synthesis of IL-1, IL-6, IL-8,        IL-12 and TNF, all proteins of a pro-inflammatory nature.    -   HAS is effective in both diminishing the synthesis of new mRNA        and in significantly reducing the protein synthesis of IL-2,        IL-7 and IL-12 on the part of the synoviocytes (for IL-12) and        cell components of the I.S., both in situations in which an        immune response is not demanded, and also in particular        inflammatory stress events in which the immune cell responds by        producing a cytokine cascade and, above all in this case, the        data presented reveal the greater inhibitory effect of HAS.    -   HAS is effective in inhibiting the binding of TNF, IL-1 and IL-6        to their receptor. These results are of fundamental importance        as they prove that the behaviour of the sulfate is completely        similar to that of specific monoclonal antibodies for the        receptors of the above pro-inflammatory proteins, consequently        capable of blocking their function even if they do not have this        specificity. This receptor blockage represents the most        effective way of antagonizing the pro-inflammatory and tumoral        effects of the TNF, IL-1 and IL-6 factor, thus opening up new        horizons in clinical experimentation, allowing the perfectioning        of new therapeutic approaches in the treatment and/or prevention        of an extremely high number of pathologies, considering the role        that TNF, IL-1 and IL-6 have in the onset and progression of        numerous diseases.

The Applicant therefore describes and claims the new use of HAS for thepreparation of a medicine:

-   -   for the prevention and/or treatment of pathologies associated        with immune deficiency and, in particular, deficiency of IL-10,        stimulating the synthesis of antiinflammatory cytokines as new        local and systemic therapy of pathologies such as vitiligo,        eczema, psoriasis and dermatitis;    -   for the prevention and/or treatment of pathologies associated        with the increase/activation of IL-1, IL-2, IL-6, IL-7, IL-8,        IL-12 and TNF;    -   for the prevention and/or treatment of asthma, rheumatoid        arthritis, and arthrosis associated with the activation of IL-1,        IL-6 and TNF;    -   for the prevention and/or treatment of pathologies associated        with endothelial damage in blood vessels;    -   for the prevention and/or treatment of skin diseases such as,        for example, dermatitis and cutaneous lymphoma;    -   for the prevention and/or treatment of diseases of an autoimmune        nature such as rheumatoid arthritis, Crohn's disease (and all        chronic intestinal inflammations), psoriasis and asthma,        diabetes mellitus, encephalomyelitis, Multiple Sclerosis, Lupus        erythematosus and for the treatment of rejection in organ        transplants;    -   for the prevention and/or treatment of neoplasias such as, for        example, leukaemia and Hodgkin's lymphoma;    -   for the prevention and/or treatment of astrogliosis,        astrocytosis and demyelination of the neuronal fibres;    -   for the prevention and/or treatment of pathologies associated        with the activation of osteoclasts, such as the osteoporosis;    -   for the prevention and/or treatment of vascular pathologies of        the arterio/atheroschlerotic type, venous thrombosis and        vasculitis, associated with the activation of TNF, IL-1 and        IL-6;    -   for the prevention and/or treatment of febrile pathologies.

The Applicant has also demonstrated, in the experimentations describedhereunder, the powerful antiviral action of HAS vs different types ofviruses:

-   -   the results obtained show the effectiveness of HAS (both degree        1 and degree 3) in inhibiting the viral replication of the HIV 1        and 2 virus. The experimentation has also demonstrated that not        all sulfated hyaluronic acids known in the state of the art act        effectively vs viral replication, as only HAS in which the        sulfation has taken place with respect to the hydroxyl groups        alone has proved to be active, where HA-NS is not effective in        blocking viral cytopathogenicity.    -   Experimental data confirm the antiviral action of HAS1 and HAS3        vs Herpes Simplex virus 1 and 2 and vs the virus of vesicular        stomatitis (VSV). The first form, extremely widespread, is        responsible for the appearance of characteristic febrile        vesicles which normally affect the facial cutis (lips,        nostrils); it is also called herpes simplex labialis. The        infection caused by lip herpes can easily reappear as the virus        survives inside the cells and is not even eliminated with the        use of effective drugs. The second form is a genital infection,        also known as herpes genitalis. Both are caught by physical and        sexual contact. Due to the location of the virions in the        nervous ganglia, where they can remain quiescent for a long        period of time, the herpetic infection has recurring        characteristics in correspondence with stressing events of the        immune system and usually reappears in the primary site. The        virus of vesicular stomatitis is an RNA-virus, it strikes        mammals (mainly animals) and is used in the laboratory for        studying the development of the life-cycle of the RNA-virus. A        comparison between HA-NS1 and HAS1 shows once again that not all        sulfated hyaluronic acids are equivalent, as HA-NS1 has proved        not to be active at all, whereas both HAS 1 and 3 show a very        strong antiviral activity vs Herpes Simplex and also vs VSV.        None of the samples tested prove to be cytotoxic towards the        host cell, the minimum cytotoxic concentration obtained, in        fact, is equal to that of reference drugs normally used in        clinical practice for the treatment of Herpes, and on an average        has proved to be 100 times higher than that revealed active in        the inhibition of viral replication.    -   Experimental data obtained for both HAS1 and HAS3 have revealed        a clear and significant antiviral result vs Cytomegalovirus:        this is a particular type of virus which enters some types of        cells of our organism in which it reproduces itself        parasitically causing their death. It belongs to the same family        as herpes labialis and herpes genitalis, chickenpox and        infective mononucleosis. Epithelial cells, mucous membranes,        lymphonodes are the site of multiple primary infection. It        remains in latent form for life in the peripheral blood, in the        epithelium of the renal tubules and in the epithelium of the        salivary glands. Serious forms are found in immunocompromised        subjects (such as those affected by AIDS and transplant subjects        in immunosuppressive therapy) involving various organs:        pneumonia, hepatitis, colitis, esophagitis, nephritis. The        treatment therapy consists in the administration of drugs such        as ganciclovir, valganciclovir and foscarnet (inhibitors of the        synthesis of viral DNA). Also in this case, HA-NS1 has proved to        be non-active in inhibiting the proliferation of the virus        confirming the absolute diversity, as antiviral capacity,        between the two types of sulfated products.

The Applicant therefore describes and claims the new use of HAS for thepreparation of a medicine:

-   -   for the prevention and/or treatment of HIV;    -   for the prevention and/or treatment of herpes simplex labialis        and herpes genitalis;    -   for the prevention and/or treatment of the virus of vesicular        stomatitis;    -   for the prevention and/or treatment of Cytomegalovirus.

Finally, the Applicant describes the preparation of variouspharmaceutical formulations/compositions containing HAS as sole activeprinciple, or in association with other pharmacologically and/orbiologically active agents such as, for example, steroids, hormones,proteins, trophic factors, vitamins, non-steroid anti-inflammatory drugs(FANS), chemotherapy drugs, calcium-antagonists, antibiotics, antiviralagents, anticoagulants and/or fibrinolytic agents, local anaesthetics,enzymes such as, for example, collagenase and/or hyaluronidase and/orother proteases; it can be formulated with polymers such as hyaluronicacid and its derivatives, carboxymethylcellulose and/or other polymersof a natural (such as collagen) or synthetic nature.

The pharmaceutical composition in question can be administeredsystemically (endovenously or arterially, intramuscularly,intraperitoneally, hypodermically or orally), it can be used for a topicapplication by dermal and/or transdermal absorption, it can beadministered by inhalation/aerosol (especially for the treatment ofasthmatic pathologies), intra-articularly or it can be administereddirectly in the site to be treated by direct injection.

The pharmaceutical composition in question can be formulated as anointment, lipogel, hydrogel, lipstick, cream, vaginal ovules andbougies, foam, mucosal gel, ophthalmic preparations, douches, mouthwash,patches for dermal and/or transdermal absorption, especially of FANS andhormones, solution for inhalant use.

Some examples of the preparation of HAS degree 1 and 3, pharmaceuticalformulations containing it, are provided for purely descriptive andnon-limiting purposes, together with the results obtained byexperimentation in vitro.

Example 1

Preparation of the Tetrabutylammonium Salt of Hyaluronic Acid (HA)Having an Average Molecular Weight Equal to 200KD (Ranging from 150,000to 250,000 Da)

5.00 g of hyaluronic acid of a fermentative sodium salt origin (200 KD)were dissolved in 250 ml of water and the resulting solution waspercolated through a glass column pre-filled with 100 cm³ of Dowex resinin the form of tetrabutylammonium (TBA). The eluted solution of HA-TBAsalt is collected and freeze-dried. 7.50 g of product are obtained.

Example 2

Synthesis of Sulfated HA Starting from HA Having an Average MolecularWeight of 200 KD and a Sulfation Degree Equal to 3 Sulfate Groups PerRepetitive Unit

Method A

10.0 g of the TBA salt of hyaluronic acid having an average molecularweight of 200 KD prepared according to Example 1, are dissolved in 300ml of dimethylsulfoxide (DMSO); 26.0 g of the complex SO₃-pyridine(sulphur trioxide and pyridine, hereafter abbreviated as PySO₃) aredispersed in 150 ml of DMSO, and then added to the solution of HA. After20 hours under mechanical stirring at a temperature of 21° C., thereaction is interrupted adding 0.1 volumes of water; the raw reactionproduct is isolated by precipitation after the addition of 2 volumes ofethanol. The solid obtained is dispersed in 150 ml of water and the pHbrought to neutrality with NaOH 1 M. The mixture is exhaustivelydialysed against water through a membrane with a cut-off of 12-14,000Da. The dialysed product is subjected to freeze-drying. 9.7 g of productare obtained with a sulfation degree equal to 3 sulfate groups perrepetitive unit (yield=88%).

Method B

32.0 g of the TBA salt of hyaluronic acid having an average molecularweight of 200 KD prepared according to Example 1, are dissolved in 900ml of N-Methyl-Pyrrolidone (NMP); 100 g of PySO₃ are dispersed in 600 mlof NMP, and then added to the solution of HA. After 20 hours undermechanical stirring at a temperature of 21±1° C., the reaction isinterrupted adding 0.5 volumes of water; The pH initially lower than2.5, is brought to neutrality with NaOH (in solution). The reaction rawproduct is isolated by precipitation by the addition of 2.5 volumes ofmethanol and washed with 2 volumes of a methanol/water mixture 8/2. Thesolid is re-dissolved and exhaustively dialysed against water through amembrane with a cut-off of 12-14,000 Da. 30.4 g of product are obtainedwith a sulfation degree equal to 3 sulfate groups per repetitive unit(yield=86%).

Example 3

Synthesis of Sulfated HA Starting from HA Having an Average MolecularWeight of 200 KD and a Sulfation Degree Equal to 1 Sulfate Group PerRepetitive Unit

Using the procedure illustrated in Example 1, 10.0 g of TBA salt of HAare prepared, which are dissolved in 350 ml of DMSO. 10.0 g of thecomplex PySO₃ are dispersed in 100 ml of DMSO, and then added to thesolution of HA. After 20 hours under mechanical stirring at atemperature of 21° C., the reaction is interrupted adding 0.1 volumes ofwater; the raw reaction product is isolated by precipitation after theaddition of 2.5 volumes of ethanol. The solid obtained is dispersed in150 ml of water and the pH brought to neutrality with NaOH 1 moles/l.The mixture is exhaustively dialysed against water through a membranewith a cut-off of 12-14,000 Da. The dialysed product is subjected tofreeze-drying. 7.54 g of product are obtained with a sulfation degreeequal to 1.0 sulfate group per repetitive unit (yield=93%).

Example 4

Synthesis of Sulfated HA Starting from HA Having a Low Molecular Weight(Average MW of 10 KD, Ranging from 10,000 to 50,000 Da) and a SulfationDegree Equal to 3 Sulfate Groups Per Repetitive Unit

Using the procedure illustrated in Example 1, 12.4 g of TBA salt oflow-molecular-weight hyaluronic acid are prepared, which are dissolvedin 300 ml of NMP. 40 g of PySO₃ are dispersed in 100 ml of NMP, and thenadded to the solution of HA. After 20 hours under mechanical stirring ata temperature of 21° C., the reaction is interrupted adding 0.5 volumesof water. The pH initially lower than 2.5, is brought to neutrality withNaOH 4M. The reaction raw product is isolated by precipitation by theaddition of 2.5 volumes of methanol and washed with 2 volumes of amethanol/water mixture 8/2. The solid is redissolved and exhaustivelydialyzed against water through a membrane with a cut-off of 3,500 Da.12.0 g of product are obtained with a sulfation degree equal to 3.0sulfate groups per repetitive unit (yield=85%).

Example 5

Synthesis of Sulfated HA Starting from HA Having a Low Average MolecularWeight and a Sulfation Degree Equal to 1 Sulfate Group Per RepetitiveUnit

Using the procedure illustrated in Example 1, 12.4 g of TBA salt of HAare dissolved in 300 ml of DMSO. 16.0 g of PySO₃ are dispersed in 100 mlof DMSO and then added to the solution of HA. After 20 hours undermechanical stirring at a temperature of 21° C., the reaction isinterrupted adding 0.1 volumes of water; the reaction raw product isisolated by precipitation by the addition of 2.5 volumes of ethanol. Thesolid obtained is dispersed in 150 ml of water and the pH brought toneutrality with NaOH 1 moles/l. The mixture is exhaustively dialyzedagainst water through a membrane with a cut-off of 3,500 Da. Thedialyzed product is subjected to freeze-drying. 9.04 g of product areobtained with a sulfation degree equal to 1.0 sulfate group perrepetitive unit (yield=90%).

Example 6

Synthesis of Sulfated HA Starting from HA Having a Molecular Weightwithin the Range of 500-730 KD Da and a Sulfation Degree Equal to 3Sulfate Groups Per Repetitive Unit

21.0 g of hyaluronic acid sodium salt of an extractive origin (500-730KD) are dissolved in 1.5 l of water and the resulting solution ispercolated through a glass column pre-filled with 450 cm³ of Dowex resinin the form of TBA. The eluted solution of HA-TBA salt is collected andfreeze-dried. 32.0 g of product are obtained, which are dissolved in1.35 l of NMP; 100 g of PySO₃ are dispersed in 650 ml of NMP, and thenadded to the solution of HA. After 20 hours under mechanical stirring ata temperature of 23±1° C., the reaction is interrupted adding 0.5volumes of water. The pH initially lower than 2.5, is brought toneutrality by the addition of NaOH (in solution at a concentration of 4moles/1). The reaction raw product is isolated by precipitation by theaddition of 2.5 volumes of methanol and washed with 3.5 volumes of amethanol/water mixture 8/2. The solid is re-dissolved and exhaustivelydialyzed against water through a membrane with a cut-off of 12-14,000Da. 30.3 g of product are obtained with a sulfation degree equal to 3sulfate groups per repetitive unit (yield=83%).

Example 7

Synthesis of Sulfated HA Starting from HA Having a Molecular Weightwithin the Range of 500-730 KD and a Sulfation Degree Equal to 1 SulfateGroup Per Repetitive Unit

21.0 g of hyaluronic acid sodium salt of an extractive origin (500-730KD) are dissolved in 1.5 l of water and the resulting solution ispercolated through a glass column pre-filled with 450 cm³ of Dowex resinin the form of TBA. The eluted solution of HA-TBA salt is collected andfreeze-dried. 32.0 g of product are obtained, which are dissolved in1.65 l of NMP; 40 g of PySO₃ are dispersed in 350 ml of NMP, and thenadded to the solution of HA. After 20 hours under mechanical stirring ata temperature of 25±1° C., the reaction is interrupted adding 0.5volumes of water. The pH initially lower than 2.5, is brought toneutrality by the addition of NaOH (in solution at a concentration of 4moles/1). The reaction raw product is isolated by precipitation by theaddition of 3.5 volumes of methanol and washed with 3.5 volumes of amethanol/water mixture 8/2. The solid is re-dissolved and exhaustivelydialyzed against water through a membrane with a cut-off of 12-14,000Da. 22.5 g of product are obtained with a sulfation degree equal to 1.0sulfate group per repetitive unit (yield=87%).

Example 8

Evaluation of the Regulatory Effect of HAS Degree 1 and Degree 3 on theGene Expression of IL-10 and IL-12 in Human Synoviocytes

Human synoviocytes previously expanded in vitro and maintained in aculture at 37° C. with a medium of DMEM containing 10% of FCS, wereseeded at a concentration of 20,000 cells per well. Sulfated HA degree 1(HAS1) and degree 3 (HAS3) prepared as described in Examples 1-3, werethen added to the culture medium at concentrations of 0.1 and 0.5 mg/ml(for both samples), whereas the control treatment is represented bynon-sulfated HA having an average molecular weight (MW) of 200 KD. After3 days of treatment, the PCR Real Time was effected to evaluate the geneexpression of IL-10 and IL-12: the cell RNA was extracted using the“Trizol” method, following the indications of the supplier (TRIZOLreagent, LIFE Technologies, GIBCO BRL). In short, the cells were lysedby the addition of 1.0 ml of Trizol and the total RNA was quantified bymeasuring its absorbance at 260 nm. The appropriate primers wereselected for each gene to be amplified, using the software Primer3(Roche Molecular Diagnostics, Pleasanton, Calif., USA). The geneexpression was evaluated by means of PCR Real Time effected with aRotor-gene TM5500 (Corbett research, Sydney, Australia). The PCRreactions were effected using primers at 300 nm and SYBR Green(Invitroge, Carlsbad, Calif., USA) at 40 cycles of 15 s at 95° C., and 1min. at 60° C. The value of Fluorescence thresholds (Ct)” wasautomatically determined by the software, evaluating an amplificationcoefficient for the genes studied between 92 and 110%. For each sampleof cDNA, the gene expression value was expressed in terms of the ratiobetween the ct of the house keeping gene (i.e. the gene for thebeta-Actin protein which represents the control gene as it is present inevery cell and is not subjected to the influence of HAS) and the ct ofthe gene of interest (i.e. the gene for IL-10 and IL-12), consequentlythe house keeping ct/gene ct value is indicated in the axis of theordinates, which therefore indicates the quantity of mRNA expressed bythe gene which is being studied. The results obtained are expressed inFIGS. 1 and 2:

FIG. 1: the treatment of human synoviocytes with HAS1 and HAS3 caused asignificant increase in the gene expression of the cytokine IL-10 vs thecontrol treated with non-sulfated HA.

FIG. 2:

Also in this experiment, both sulfation degrees (degree 1 and degree 3)of HAS proved to be capable of significantly reducing the geneexpression of IL-12, halving the synthesis of its mRNA vs the controltreated with non-sulfated HA. The sulfated hyaluronic acid thereforeproved to be:

-   -   capable of stimulating the production of new mRNA for the        synthesis of anti-inflammatory cytokines, thus increasing the        defence capacity of the cell and consequently of the whole        organism, vs those pathologies previously described in which        IL-10 proved to be of fundamental importance for the resolution        and/or improvement of diseases such as asthma, rheumatoid        arthritis, arthrosis and all inflammations in which IL-10 is        involved.    -   effective in diminishing the synthesis of new mRNA of the highly        pro-inflammatory cytokine IL-12, proving to be a valid        anti-inflammatory agent capable of intervening on the expression        of proteins involved in the pathogenesis of invalidating        diseases such as psoriasis, arthritis and all those previously        described.

Example 9

Inhibition of the Binding of TNF to its Receptor Expressed in MonocyteLines: Evaluation of the Effectiveness of HAS Degree 1 and Degree 3 atDifferent MW Values

These experiments were effected to evaluate the effectiveness of thesamples tested (prepared according to Examples 1-4) on the capacity ofinhibiting the binding of TNF to its receptor expressed by cells of theI.S. normally used in vitro for this type of experiment, carried outwith iodinated cytokine components for an evaluation in RadioligandBinding assays.

The experimental procedure was effected as described in Baglioni C. etal., J Biol Chem, 1985, 260:13395-13397.

In short, the line of human histiocytes of the lymphoma U937 was used,with characteristics of monocytes sensitive to the cytotoxic activity ofTNF, expressing its relative receptor. The cells were initiallyincubated with ¹²⁵I-TNF 0.028 nM (carried in water) contemporaneouslywith the samples to be analyzed (at a concentration of 1 mg/ml whichproved to be the lowest concentration which causes the maximuminhibition), in an incubation buffer consisting of 50 mM Tris-HCL pH7.4, 0.5 mM EDTA, at 4° C. for 3 hours.

At the end of the incubation, the cells were centrifuged withdibutylphthalate/dinonylphthalate 2/1 and the pellet obtained wascounted in a γ-counter.

The results obtained are expressed in FIG. 3:

The results obtained show the effectiveness of HAS in totally (100%)inhibiting the binding of TNF to its receptor, for both degree 1 anddegree 3, with a medium and low MW. These results are of fundamentalimportance as they prove that the behaviour of the sulfated product iscompletely analogous to that of a monoclonal antibody specific for theTNF receptor, capable therefore of blocking its function. This receptorblockage consequently represents the most effective way of antagonizingthe pro-inflammatory and tumoral effects of the TNF factor.

Example 10

Inhibition of the Binding of the Cytokine IL-1 to its Receptor Expressedin Fibroblast Lines: Evaluation of the Effectiveness of HAS Degree 3 atDifferent MW Values

These experiments were effected to evaluate the effectiveness of thesamples tested (prepared according to Examples 1-3 and 4) on thecapacity of inhibiting the binding of IL-1 to its receptor expressed by3T3 cells of mice, normally used in vitro for this type of experiment,carried out with iodinated cytokine components for an evaluation inRadioligand Binding assays.

The experimental procedure was effected as described in Chin J et al., JExp Med, 1987, 165:70-86.

In short, the line of murine fibroblasts 3T3 was used, sensitive to thecytotoxic activity of IL-1, expressing its relative receptor. The cellswere initially incubated with ¹²⁵I-IL-1 10 pM (carried in water)contemporaneously with the samples to be analyzed (at a concentration of1 mg/ml which proved to be the lowest concentration which causes themaximum inhibition), in an incubation buffer consisting of RPMI 1640containing 20 mM HEPES pH 7.2 and 1% BSA, at 37° C. for 2 hours. At theend of the incubation, the cells were washed with phosphate buffer, thendissolved in 2.5 M of NaOH and counted in a 7-counter.

The results obtained are expressed in FIG. 4:

The results obtained show the effectiveness of HAS (with both medium andlow MW) in inhibiting the binding of IL-1 to its receptor by 30%. Theseresults are extremely significant as they prove that the behaviour ofthe sulfated product is completely analogous to that of a monoclonalantibody specific for the receptor of the cytokine in question, capabletherefore of blocking its function. This receptor blockage representsthe most effective way of antagonizing the pro-inflammatory and tumoraleffects of IL-1, as previously described.

Example 11

Inhibition of the Binding of the Cytokine IL-6 to its Receptor Expressedin Human Myeloma Cells: Evaluation of the Effectiveness of HAS Degree 3at Different MW Values

These experiments were effected to evaluate the effectiveness of thesamples tested (prepared according to Examples 1-3 and 4) on thecapacity of inhibiting the binding of IL-6 to its receptor expressed inhuman myeloma U266, normally used in vitro for this type of experiment,carried out with iodinated cytokine components for an evaluation inRadioligand Binding assays.

The experimental procedure was effected as described in Taga T. et al.,J Exp Med, 1987, 166:967-981.

In short, the line of human myeloma U266 was used, sensitive to thecytotoxic activity of IL-6, expressing its relative receptor. The cellswere initially incubated with ¹²⁵I-IL-6 0.08 nM (carried in water)contemporaneously with the samples to be analyzed (at a concentration of1 mg/ml which proved to be the lowest concentration which causes themaximum inhibition), in an incubation buffer consisting of RPMI 1640containing 25 mM HEPES pH 7.1 and 10% BSA, at 4° C. for 16 hours. At theend of the incubation, the cells were washed with phosphate buffer,centrifuged at 9,000 rpm and the pellet counted in a γ-counter.

The results obtained are expressed in FIG. 5:

The results obtained show the effectiveness of HAS, with both medium andlow MW, in inhibiting the binding of IL-6 to its receptor by up to 100%.These results consequently prove that the behaviour of the sulfatedproduct, also in this case, is completely analogous to that of amonoclonal antibody specific for the receptor of the cytokine inquestion, capable therefore of blocking its function. This receptorblockage represents the most effective way of blocking thepro-inflammatory effects of IL-6.

Example 12

Evaluation of the Inhibitory Effect of HAS Degree 1 and Degree 3 on theProtein Synthesis of the Cytokines IL-2, IL-7, IL-10 and IL-12 in HumanPBMC

For these experimentations, mononucleate cells of human peripheral blood(PBMC) were adopted, deriving from many different donors for evaluatingthe effect of HAS on the production of the cytokines listed above,using:

-   -   non-sulfated HA (average MW: 200 KD),    -   HAS1 and HAS3 (prepared as described in Examples 1-3 (average        MW: 200KD).

The separation of the PBMC (Bøyum A., Scand J Clin Lab Invest 21 Suppl,1968, 97:77-89) was effected using the product Ficoll-Paque PLUS (GEHealthcare) and following the protocol indicated by the supplier. At dayzero 100,000 cells were seeded per well (using plates with 96 wells) in200 μl of medium RPMI 1640, to which 10% of fetal bovine serum, HEPES 10mM, Glutamine 2 mM, 1% Penicillin-Streptomycin 100 U/ml, had been added.The effect of all the samples was evaluated on non-treated PBMC withagents which stimulate the synthesis of cytokines, or stimulated withLipopolysaccharide LPS (10 μg/ml) (highly pro-inflammatory) or withPhytohemagglutinin PHA (10 μg/ml) (a substance capable of stimulatingthe lymphocytes to divide themselves), both agents capable ofstimulating the synthesis of cytokines. The cells were treatedseparately with the three compounds at a concentration of 0.1 mg/ml or 1mg/ml. After 24 hours of incubation at 37° C. (5% CO₂), 100 μl ofsupernatant were taken from each well in order to analyze the productionof IL-2, IL-7, IL-10 and IL-12.

The quantification of the inflammation mediators was effected by meansof the SearchLight® technology, using a Custom Human 9-Plex Array platefollowing the protocol indicated by the supplier in the technical card.The results obtained are expressed in FIGS. 6-9: These graphs clearlyshow that HAS degree 1 and degree 3 are capable of significantlyreducing the synthesis of IL-2, IL-7 and IL-12 on the part of monocytes,both when the cells are not stimulated and also when, on the contrary,they are stimulated by specific and powerful inflammatory factors and/ormitogens. HAS therefore proves to be a molecule with precisepharmacological characteristics, capable of modulating/regulating thesynthesis of cytokines with a marked anti-inflammatory activity, both insituations where an immune response is not stimulated, and in particularinflammatory stress events in which the immune cell responds byproducing a cytokine cascade and, above all, in this case, the datapresented reveal a greater HAS modulating effect.

FIG. 9, on the other hand, confirms the evident stimulus for theproduction of IL-10 also for cells such as human monocytes (besidessynoviocytes) belonging to the Immune System. It is consequently againconfirmed that HAS is capable of modulating the synthesis of cytokines,stimulating those which are anti-inflammatory and inhibiting thesynthesis of pro-inflammatory cytokines.

Example 13

Evaluation of the Antiviral Action of HAS Degree 1 and Degree 3 VsHA-NS:

HIV-1 (III_(B)) and HIV-2 (ROD) Virus

The activity of the samples tested was determined by evaluating theinhibition of the cytopathogenicity determined by the HIV-1 virus(obtained from T lymphocytes infected by the type III_(B) virus) andHIV-2 virus (obtained from T lymphocytes infected by the type ROD virus)in MT-4 cells. The experimental procedure was effected as described inBaba M. et al., ANTIMICROB. AGENTS CHEMOTHER., 1988, 32:1742-1745.

In short, human T lymphocytes called MT-4 infected by the HIV-1 virusstrain III_(B) and HIV-2 strain ROD, were used for this assay. Differentconcentrations of the samples tested were adopted for this test, usingdextransulfate as positive control, a polysaccharide known for itscapacity of inhibiting different types of viruses. The samples testedwere:

-   -   1. HS-NS1: sulfated hyaluronic acid prepared by sulfation of the        glucosamine after deacetylation, degree 1 (EP0971961), to        compare it with    -   2. HAS1: sulfated hyaluronic acid only in its hydroxyl groups,        degree 1, and    -   3. HAS3: sulfated hyaluronic acid only in its hydroxyl groups,        degree 3, prepared as described in Examples 1-3 (all the samples        with average MW: 200KD).

The antiviral activity of the samples tested is expressed as the minimumconcentration required for inhibiting the cytopathogenicity of the virusby 50%: IC50. The cells were treated with various concentrations of theabove samples added immediately after the infection with infective dosesof HIV. After 5 days at 37° C., the number of vital cells is determinedwith the MTT test: tetrazole salt subjected to redox reaction only bythe mitochondrial enzymes of vital cells. The infected and non-treatedcells lose the progressive capacity of transforming the tetrazole saltto formazane, consequently, if these cells keep this capacity intactafter infection and treatment, this means that this treatment hasinhibited the viral action, thus blocking the pathogenicity of the virus(Dezinot F. et al., J Immunol. Methods, 1986, 22(89): 271-277).

The results obtained are expressed in FIG. 10:

The results obtained demonstrate the effectiveness of HAS (both degree 1and degree 3) in inhibiting the viral replication of the virus HIV 1 and2, in a way which is completely comparable to the positive control(represented by the polysaccharide dextran-sulfate). The experimentationalso demonstrated that not all sulfated hyaluronic acids known in thestate of the art act effectively vs the replication of the virusstudied, as the Applicant has proved that only the HAS whose sulfationhas taken place with respect to its hydroxyl groups proves to be activein its powerful antiviral action as HA-NS is not effective in blockingthe viral cytopathogenicity.

Herpes Simplex Virus-1, Herpes Simplex Virus-2, Vesicular StomatitisVirus

The activity of the samples tested was determined by evaluating theinhibition of the cytopathogenicity caused by the virus of HerpesSimplex Virus-1, (HSV-1: KOS, F and McIntyre strain) and by the virus ofHerpes Simplex Virus-2, (HSV-2: G, 196 and Lyons strain) in E₆SMfibroblast cells which derive from muscular/embryonic skin tissue.Furthermore, the anti-viral activity was tested again vs E₆SM cellsinfected by the virus of vesicular stomatitis (Vesicular stomatitisvirus: VSV). HSV-1 is the virus which preferentially infects the oralmucous, whereas HSV-2 attacks the genital mucous. The experimentalprocedure was effected as described in Baba M. et al., ANTIMICROB.AGENTS CHEMOTHER., 1988, 32:1742-1745.

In short, confluent cell cultures were exposed to infective doses of theviruses listed above in the presence of the samples HS-NS1 (EP0971961),HAS1 and HAS3 prepared as described in Example 1 and 2 (all the sampleswith an average MW: 200KD). After an incubation period of 1 h at 37° C.,the culture medium was substituted with fresh medium only containing thesamples to be tested. The cytopathogenicity of the virus was tested onthe 2^(nd) day of incubation. The measurement of the inhibition of theviral cytopathogenicity was evaluated by determining the inhibition ofthe synthesis of DNA and RNA in the “infected” cells and subjected totreatment as indicated above: the cells were seeded in micro-wells in aculture medium containing different concentrations of the samples to betested with 2.5 μCi of 3H-thymidine and 3H-uridine per ml. After 16 h at37° C., the cells were treated with Trichloroacetic acid, washed inethanol, left to dry and counted in 7.5 ml of liquid by scintillation.The antiviral activity of the samples tested is expressed as the minimumconcentration required for inhibiting the cytopathogenicity of the virusby 50%: IC50. Furthermore, in order to also evaluate the cytotoxicity ofthe samples tested, the minimum concentrations necessary for causingmorphological damage (observable with an optical microscope) to thecells used, were determined. The comparison was effected vsdextran-sulfate and the drug Acyclovir (both molecules with a knownantiviral effectiveness, therefore used as positive control).

The results obtained are expressed in FIG. 11:

The experimental data confirm what was seen for HIV, i.e. the powerfulantiviral action of both HAS1 and HAS3: a comparison between HA-NS1 andHAS1 shows that not all sulfated hyaluronic acids are equivalent asHA-NS1 did not prove to be active, and this difference in efficacy doesnot depend on the molecular weight or the sulfation degree of hyaluronicacid, it therefore lies in the very structure of HA-NS1 vs HAS1, i.e. onthe sulfation. HAS in fact shows an efficacy equal to that ofdextransulfate and comparable to that of acyclovir, a reference drug forthe treatment of Herpes Simplex. Furthermore, it should be pointed outthat acyclovir is inactive vs VSV, whereas HAS degree 1 and 3 has a verypowerful antiviral activity vs VSV.

All the samples tested are non-cytotoxic towards the host cell, theminimum cytotoxic concentration obtained is in fact equal to that of thereference drugs normally used in clinical practice for the treatment ofHerpes, and proved to be on an average 100 times higher than that provedactive in the inhibition of the viral replication.

Cytomegalovirus

The activity of the samples tested was determined by evaluating theinhibition of the cytopathogenicity determined by Cytomegalovirus (CMV:AD-169 and Davis strain) using the previous protocol. The antiviralactivity was tested vs HEL cells (pulmonary embryonic cells) and wasexpressed as the concentration required for inhibiting the number ofplaques formed by the above virus by 50%. The results obtained areexpressed in FIG. 12:

the table indicates the clear and significant positive result obtainedfor both HAS1 and HAS3 which again confirms them as effective antiviralagents. Also in this case, HA-NS1 did not prove to be active ininhibiting the proliferation of the virus, confirming the absolutediversity between the two types of degree 1 sulfated product in havingan antiviral capacity.

Example 14

Preparation of a Formulation in the Form of a Solution for InhalationsContaining Sulfated Hyaluronic Acid Degree 1

40 mg (or 20 mg if the HAS has a MW of 500-730KD) of sulfated hyaluronicacid degree 1, having a low or medium MW, are introduced into a 50 mlglass flask, after which 15 ml of PBS 0.2M at sterile pH 7.4, are added.The mixture is subjected to stirring for about 30 minutes, until thecomplete dissolution of the powder. When the complete dissolution hasbeen obtained, 2 ml of glycol propylene and further PBS 0.2M at sterilepH 7.4 are added until the total volume of 20 ml is reached. Thesolution is maintained under stirring for a few minutes.

Example 15

Preparation of a Formulation in the Form of a Solution for InhalationsContaining Sulfated Hyaluronic Acid Degree 3

100 mg of sulfated hyaluronic acid degree 3 obtained from HA 200KD areintroduced into a 50 ml glass flask, after which 15 ml of PBS 0.2M atsterile pH 7.4, are added. The mixture is subjected to stirring forabout 30 minutes, until the complete dissolution of the powder. When thecomplete dissolution has been obtained, 2 ml of glycol propylene andfurther PBS 0.2M at sterile pH 7.4 are added until the total volume of20 ml is reached. The solution is maintained under stirring for a fewminutes.

Example 16

Preparation of a Formulation in the Form of an Injectable Solution forIntra-Articular Use Containing Sulfated Hyaluronic Acid Degree 1

500 mg of sulfated hyaluronic acid degree 1 obtained from HA with a MWof 500-730KD are introduced into a 50 ml glass flask, and PBS 0.2M atsterile pH 7.4, is then added until the total volume of 20 ml isreached. The mixture is subjected to stirring for about 60 minutes,until the complete dissolution of the powder.

Example 17

Preparation of a Formulation in the Form of a Hydrophilic Gel ContainingHAS, HA and CMC

Methyl- and propyl-parabene are dissolved in purified water at 80° C.After cooling the solution to room temperature, sodium hyaluronate isadded under stirring until dissolution followed by HAS1 (or HAS3),maintaining the stirring until complete dissolution. Glycerol andpropylene glycol are then added under stirring until completedissolution. Sodium carboxymethylcellulose (CMC) is finally added andthe mixture is mixed until a gelified solution is obtained.

Example 18

Preparation of a Formulation in the Form of a Hydrophilic Gel forMucosal Use (without Preservatives) Containing HAS and HA

Sodium hyaluronate is dissolved under stirring, and then HAS1 (or HAS3)in a quantity of water of about 90% of that envisaged in the formula.Propylene glycol, Symdiol 68 are added followed by MP Diol Glycol andthe mixture is mixed until the complete dissolution of the variouscomponents. Carbomer 974P is subsequently added and the stirring ismaintained until the homogeneous dispersion of the latter. Beads ofsodium hydroxide are dissolved in the remaining 10% of water and thissolution is added to the that previously obtained, to obtain thegelification of the aqueous phase.

Example 19

Preparation of a Formulation in the Form of a Lipstick Containing HASand HA

The correct quantity of liquid paraffin indicated in the manufacturingformula is charged into a suitable container. It is heated to 88-92° C.and white soft paraffin, hard paraffin, white beeswax, ceresin, arlacelare then added under stirring, the stirring is maintained until thecomplete melting of the various components. All-raca-tocopheryl acetate,allantoine, butylhydroxytoluene, propyl p-hydroxybenzoate are thenincorporated and the mixture is mixed until complete dissolution,maintaining the mass at 88-92° C.

The quantity of purified water envisaged in the formula is chargedseparately into a suitable container, sodium hyaluronate, HAS1 (o HAS3)are then added under stirring until complete dissolution, followed byDisodium Edetate maintaining the stirring until dissolution.

The aqueous phase is transferred under stirring to the containercontaining the molten mass, maintaining the system at 88-92° C. and thestirring until a limpid solution is obtained. The two aromatizing agentsare then added under stirring and the mixture is mixed for 10′. Themolten mass is poured into moulds and is immediately cooled to T<0° C.until solid sticks are obtained.

Example 20

Preparation of a Formulation in the Form of Vaginal Ovules ContainingHAS and HA

The gelatine is allowed to swell in 70% of purified water at 85° C.;sodium hyaluronate and HAS1 (or HAS3) are dissolved in the remainingquantity of water and this solution is mixed with the glycerine broughtto the same temperature. The glycerine solution is added to the swollengelatine solution and the stirring is maintained until the completedissolution of the gelatine. The mass is poured into moulds and cooledto T<0° C. until solid ovules are obtained.

Example 21

Preparation of a Formulation in the Form of a Hydrophilic CreamContaining HAS and HA

The oil phase is prepared by melting liquid paraffin, stearic acid andTefose 1500 under stirring at 50° C. The aqueous phase is preparedseparately by the initial dissolution at 80° C. of methyl-parabene andsubsequent cooling to room temperature and the incorporation ofglycerol, sodium hyaluronate and subsequently HAS1 (or HAS3) understirring until the complete dissolution of the various components.

The aqueous phase is joined to the oil phase and emulsification iseffected, the emulsion 0/A obtained is cooled under stirring to roomtemperature.

Example 22

Preparation of a Formulation in the Form of an OINTMENT Containing HAS

The base ointment is prepared by melting light liquid paraffin and whiteVaseline under stirring at 70° C.

After cooling to room temperature, HAS1 (or HAS3) is incorporated understirring and the mixture is mixed until a homogeneous suspension isobtained.

Example 23

Preparation of a formulation in the form of CAPSULES (hard gelatine)containing HAS

HAS1 (HAS3) is mixed with calcium phosphate, magnesium stearate andsilica by progressive dilution. The capsules are then filled.

Example 24

Preparation of a Formulation in the Form of Tablets Containing HAS

HAS1 (or HAS3) is subjected to wet granulation in a fluid bed with aligand solution consisting of water and about 70% of the total quantityof sodium CMC. The granulate obtained is subjected to sieving on an 0.8mm net.

The remaining ingredients (calcium phosphate, microcrystallinecellulose, sodium CMC and silica) are mixed and sieved on an 0.8 mm net.

The granulate previously obtained is mixed with the mixture consistingof the remaining ingredients (extragranules), and is finally mixed withmagnesium stearate (previously sieved on an 0.8 mm net), followed bycompression.

Example 25

Preparation of a Formulation in the Form of an Injective SolutionContaining HAS

After the preparation (at room temperature) of a physiological solutionbuffered at pH 6.4-7.2, lactose is dissolved under stirring and finallyHAS1 or HAS3. The solution thus obtained is filtered on 0.22 microns.

TABLE HYDROPHILIC GEL (Example 17) Components Quantity (mg/1 g ofhydrogel) HAS1 (HAS3) 40 mg (10 mg) CMC   20 mg Glycerol   100 mgPropylene Glycol 66.75 mg Sodium Hyaluronate    2 mg Methylp-hydroxybenzoate    2 mg Propyl p-hydroxybenzoate  0.2 mg PurifiedWater q.s to 1 g HYDROPHILIC GEL for mucosal use (Example 18) ComponentsQuantity (mg/1 g of hydrogel) HAS1 (HAS3)   10 mg Carbomer 974P   15 mgPropylene Glycol   100 mg Sodium Hydroxyde  0.33 mg Sodium Hyaluronate   2 mg MP-Diol Glycol  37.5 mg SymDiol 68   90 mg Purified Water q.s to1 g LIPSTICK (Example 19) Components Quantity (mg/1 g lipstick) HAS1(HAS3) 30 mg (10 mg) Liquid Paraffin 253.2 mg White Soft Paraffin 326.2mg Hard Paraffin 144.3 mg Beeswax white   96 mg Ceresin  28.2 mg Arlacel582  95.8 mg Sodium Hyaluronate    2 mg Allantoin  1.1 mg all-rac-aTocopheryl Acetate  1.1 mg Propyl p-hydroxybenzoate  0.4 mgButylhydroxytoluene  0.4 mg Purified Water  19.2 mg Disodium Edetate 1.1 mg Vanilla Flavour  0.5 mg Sweet Flavour  0.5 mg VAGINAL OVULES(Example 20) Components Quantity (mg/1 g of ovule) HAS1 (HAS3)   10 mgGlycerin   580 Gelatin   200 Sodium Hyaluronate    2 Purified Water q.sto 1 g HYDROPHILIC CREAM (Emulsion O/A) (Example 21) Components Quantitymg/1 g of cream) HAS1 (HAS3)   10 mg Tefose 1500   110 mg Glycerol   80mg Stearic Acid   33 mg Sodium Hyaluronate    2 mg Liquid Paraffin   40mg Methyl p-hydroxybenzoate    1 mg Purified Water q.s to 1 g OINTMENT(Example 22) Components Quantity (mg/1 g of ointment) HAS1 (HAS3)   20mg Light Liquid Paraffin   200 mg White Petrolatum q.s to 1 g CAPSULES(Hard Gelatine) (Example 23) Components Quantity (mg/capsule) HAS-1(HAS3) 80 mg (40 mg) Calcium Phosphate   256 mg Magnesium Stearate    7mg Silica    7 mg TABLETS (Example 24) Components Quantity (mg/tablet)HAS-1 (HAS-3)  120 mg Calcium Phosphate 200 Microcrystalline cellulose185 Sodium Carboxymethylcellulose 10 Magnesium Stearate 10 Silica 15INJECTIVE FORMULATIONS (Example 25) Components Quantity (mg/ml of sol)HAS-3   50 mg Lactose  0.93 mg Potassium Phosphate Dibasic 0.36Potassium Phosphate Monobasic 0.23 Sodium Chloride 9The description being thus described, these methods can obviously bemodified in various ways. These modifications should not be consideredas diverging from the spirit and perspectives of the invention and allthe modifications which can appear evident to a skilled person in thefield are included in the scope of the following claims.

The invention claimed is:
 1. A method for the treatment in a patient of(a) asthma associated with the activation of IL-1, IL-2, IL-6, IL-7,IL-8, IL-12 and TNF, or (b) HIV, herpes simplex labialis, herpesgenitalis, virus of vesicular stomatitis, or Cytomegalovirus; comprisingadministering to a patient in need thereof as the active agent sulphatedhyaluronic acid (HAS) being prepared starting from a HA having amolecular weight within one of the following ranges from 10,000 to50,000 Da, 150,000 to 250,000 Da and 500,000 to 750,000 Da and asulphation degree equal to 1 or
 3. 2. The method according to claim 1,wherein said administration is systemic administration.
 3. The methodaccording to claim 1, wherein said administration is topical orinhalation administration.